/ . Embryol exp. Morph. Vol. 64, pp. 321-332, 1981
Printed in Great Britain © Company of Biologists Limited 1981
321
A general cell marker for clonal analysis of
Drosophila development
By PETER A.LAWRENCE
From the MRC Laboratory of Molecular Biology, Cambridge
SUMMARY
The mitochondrial enzyme, succinate dehydrogenase, can be localized by specific histochemical stains. A naturally occurring variant which gives heat-labile enzyme is used to map
a gene responsible for succinate dehydrogenase. New alleles at the sdh locus are produced
by mutagenesis and most of these are found to be homozygous lethal to flies. However,
clones of cells which are homozygous for the new alleles can be produced by mitotic recombination, and are found to develop normally. If the chromosome arm bearing an sdh
allele also carried brown, clones can be found in the eye. At the border of the clone the
brown and sdh phenotype coincide showing that the sdh phenotype is expressed as a cell
autonomous marker. Clones of sdh have been observed in the adult epidermis, the muscles,
the gut, the heart and oenocytes where they display a clear phenotype. This new marker
may be useful in studying the development of the internal organs of Drosophila.
INTRODUCTION
In Drosophila, methods of labelling cells with indelible genetic markers
(Sturtevant, 1929) have improved considerably over recent years (GarciaBellido & Dapena, 1974; Morata & Ripoll, 1975). Typically, developing
larvae heterozygous for a marker mutation are irradiated with X-rays and,
occasionally and at random, clones of homozygous cells are produced following
somatic recombination (Stern, 1936; Becker, 1957). These experiments have
led to a description of the cell lineage of several organs of the adult fly (GarciaBellido, Ripoll & Morata, 1973, 1976; Steiner, 1976; Morata & Lawrence,
1979; Struhl, 1977). Some new principles have emerged from these studies, the
most important being the compartment hypothesis (Garcia-Bellido et al. 1973;
Crick & Lawrence, 1975).
Because the best genetic markers affect the derivatives of the adult epidermis,
such as cuticle, bristles, and pigment cells of the eye, our understanding of cell
lineage is mainly limited to parts of the adult integument. Attempts to study the
cell lineage of the internal organs of both adults and larvae have depended on
a less satisfactory set of cell markers (reviewed in Hall, Gelbart & Kankel,
1976). The most useful have been mutations affecting the activity of the enzyme
1
Author's address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge,
CB2 2QH, U.K.
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P. A. LAWRENCE
aldehyde oxidase (Janning, 1972; Szabad, Schupbach & Wieschaus, 1979), but
even this enzyme cannot be detected in all tissues.
There is therefore a need for a general-purpose cell marker which would
clearly mark each cell in every tissue without affecting development. Such a
mutation would allow the study of cell lineage of internal organs and could
lead to a description of the role of homoeotic genes in the nervous system,
mesoderm and endoderm. Here I describe the isolation of alleles at a locus
affecting the activity of succinate dehydrogenase. The mutations are recessive,
viable in cells when homozygous and give a clear cell-autonomous phenotype in
those tissues so far examined.
MATERIAL AND METHODS
Succinate dehydrogenase was chosen for two main reasons: first, specific
histochemical methods are available for material to be studied with both the
light and electron microscopes (see Pearse, 1972; Lewis & Knight, 1977).
Second, the enzyme is located in the mitochondria, and the stain precipitates
mainly in those organelles. This is important because, even in heavily stained
preparations, it is possible to see nuclei and cell boundaries in sections or whole
mounts.
The strategy involved seven steps: (1) identification of a heat-labile variant of
the enzyme succinate dehydrogenase; (2) mapping of the locus responsible for
the variant; (3) mutagenesis of wild-type chromosomes to produce new mutations at the same locus; (4) 'cleaning' of the mutagenised chromosomes;
(5) testing for cell viability of the new mutations; (6) developing staining
methods for clones of cells homozygous for the new mutations; (7) testing the
new mutations for temperature sensitivity.
(1) Identification of a heat-labile variant
The indirect flight muscles of Drosophila contain large quantities of succinate dehydrogenase and a single cut with fine scissors removes a piece of cuticle from the notum containing a chunk of flight muscle. This can then be floated on hot Ringer solution (Robb, 1969)
for a given period and tested for enzyme activity by transfer to a staining solution containing
the substrate sodium succinate and the dye nitroblue tetrazolium. For details, please see
section 6 of these methods.
The activity of succinate dehydrogenase is seen as a blue precipitate. In wild-type muscles
of Canton S, Oregon R and many other strains the enzyme becomes inactivated after 15 min
at 55 °C, but is still active after heating for 15 min at 54 °C. A collection of 85 strains, all independently isolated from the wild, were therefore screened at 53 and 55 °C to look for variants
in heat lability. Only one variant was found; the succinate dehydrogenase activity disappeared
after heating the piece of thorax at 53°Cfor 15 min, but survived heating at 52°C. It was named
sdh1. This stock, C154, was originally isolated at Bonake in the Ivory Coast by G. de Jong
and was kindly provided by the Department of Genetics, University of Cambridge.
A general cell marker for clonal analysis of Drosophila
323
(2) Mapping of the sdh1 variant
The heat-sensitive phenotype segregated with the second chromosome, and was first
located on the right arm between curved (2-15) and brown (2-104.5). It was positioned more
accurately at 2-89± 1 (95% confidence limits) by staining 199 recombinants between M(2)S7
(2-77.5) and brown. (For genetic nomenclature see Lindsley & Grell, 1968.)
(3) Mutagenesis
bpr adult males were fed with ethylmethane sulphonate (Lewis & Bacher, 1968) and crossed
to en sdh1 bw females. Some of these males were crossed to attached-X females to make an
independent measure of the induced mutation rate. The sex ratio of their offspring, when
compared to controls, indicated a lethal hit rate of about 0-7 per X-chromosome. We took
about 3000 Fl males, each was then given the company of two en sdh1 bw females. Of these
individual matings, 2345 produced larvae and subsequently the Fl males were taken and
their flight muscles heated at 53 °C for 12 min and stained for succinate dehydrogenase. These
were processed in batches of ten with // stw sdh1 and y/ 36a , sdh+ muscles as controls. If all
ten experimental and the wild-type muscle chunks stained blue and the sdh1 controls were
unstained, all ten tubes were discarded. If one or more failed to stain the batch of tubes was
kept and eight F2 en sdh1 bw/bpr flies heated and stained from each tube (with appropriate
controls). If all eightfliesfrom one tube failed to stain, that tube was tested again on another
day. If once again the muscle chunks failed to stain males were outcrossed to make a. bpr
sdh*/CyO,pr en2 stock (where sdh* indicates a possible mutant allele of sdh). From the
2345 chromosomes screened in this way eleven putative mutations were isolated. In addition,
six tubes generated indirect flight muscles that stained pale blue which indicated partial
enzyme activity; these have not been analysed further. The eleven putative alleles were
crossed inter se and six were found to be lethal in trans with each other, implying that sdh is
a locus in which mutations can be lethal; these six mutations were numbered sdhz-sdh%. One
other putative allele (sdh2) was viable in trans with all six lethals givingflieshaving heat-labile
succinate dehydrogenase. The muscles of thesefliesstained positively after heating at 50 °C for
15 min but not after heating at 51 °C. sdh2 is located on a chromosome associated with
a dominant female sterile phenotype. The other four putative alleles were discarded as they
were viable in most combinations, and when in trans with the lethal alleles of sdh yielded
flies with heat-stable succinate dehydrogenase. Dr Geoff Richaids kindly examined salivary
chromosomes of sdh2, sdh3, sdh6 and sdhs, and detected no aberrations.
(4) 'Cleaning'' of the chromosomes
In case the mutagenised chromosomes carried lesions at other loci than sdh, the left arm of
the second chromosome was completely replaced and the right arm was partially substituted
by making two separate recombinations between cinnabar and brown. This was done for
sdh3, sdh\ sdh\ sdh6 and sdh*.
(5) Cell viability
While chromosome cleaning was under way, the alleles sdh2, sdh3, sdh*, sdh5, sdh6 and sdh8
were tested to see if they were viable in homozygous clones of cells. The Minute technique
(M'orata & Ripoll, 1975) was used and the Minute itself scored as a bristle marker. Usually,
y; bpr sdhfCyO, pr en2 females were crossed to DfscH<i, y~; M(2)S7sdh+ Dpsc*2, y+/CyO,
pr en2 males; the progeny were X-irradiated as larvae with 1000R (for conditions, see
Lawrence & Morata, 1977). Fl femalesy/DfscS2, y—, bpr sdh/M(2)S7sdh+ Dpsc™, y+ were
collected and thoraces and legs mounted for scoring under the compound microscope
(Dfse^ refers to the first chromosome, and DpscS2 to the second chromosome of TO ; 2)scs2).
Because M(2)S7 is proximal and DpscS2 distal to sdh, ye/low Minute clones are either sdh or
sdh{/sdh, but yellow Minute+ clones are all homozygous for sdh. The Minute phenotype
can be reliably scored in the bristles of the leg, sternopleura and notum. In all four mutants
324
P. A. LAWRENCE
tested, the yield of yellow Minute* clones was high, showing that all these six sdh alleles are
cell viable. The clones are large, normal in phenotype and not noticeably different from
control sdh* clones made by irradiation at the same time.
(6) Staining methods
Staining methods were developed initially in tissues which could be independently marked the adult epidermis and the eye. For the adult epidermis, thoraces containing large yellow
Minute* sdh clones are dissected so that the epidermis is exposed to the staining solution and
left for an hour or so. The staining solution, adjusted to pH 70-7-2, consists of five parts each
of 4 mg/ml nitroblue tetrazolium, 0-2 M Tris at pH 7-4,002 M magnesium chloride, 1 M sodium
succinate and two parts each of 01 M sodium azide and 01 M sodium cyanide (Pearse, 1972).
We do not usually make separate solutions of the cyanide, azide and succinate as recommended by Pearse. Small aliquots of the complete solution are kept frozen and continue to
give satisfactory staining after several weeks at -20°C. A control solution, in which the
succinate is replaced by malonate, is also useful; if there is blue precipitate after staining in
this control solution it must be due to enzymes other than succinate dehydrogenase (Pearse,
1972). Staining is usually carried out in the dark in a few millilitres of solution at room
temperature, the pieces of fly frequently floating on the surface. Following staining the
preparations are dehydrated in alcohol,fixedin Carnoy's solution (5-10 min), cleared in oil
of cedarwood and mounted in Euparal.
In the adult epidermis the clones are visible as a pale-blue area in a dark-blue surround,
and there is a sharp interface between the two areas so that individual cells can be classified
as pale blue or dark blue. This interface coincides closely with the boundary between yellow
and yellow* bristles. To assess clones in the eye, en sdh8 bw/M(2)S7 or en sdh6 bw/M(2)c33a
larvae were irradiated. Eyes bearing brown clones were found down the dissecting microscope
and the heads cut in half, these bisected heads were stained for 1-4 days and then fixed in
half-strength Karnovsky's solution, dehydrated in acetone, embedded in Araldite, and
sectioned at 1-4 /tm (Lawrence & Green, 1979). There was a striking difference between the
staining of bw sdh and bw+ sdh* territories in the eye, but there was still a pale blue background in the mutant clone (Fig. 3). With help from Michael Wilcox we found the simplest
way to remove this staining within the clone is to gently heat-fix the material. The tissue is
heated in Drosophila Ringer solution. The conditions must be carefully controlled; we used
a waterbath and measured the temperature with an accurately calibrated thermometervariation in most normal laboratory thermometers may be too great. The optimal temperature
has to be worked out by trial and error as some reduction in the succinate dehydrogenase
activity outside the clone also occurs. For example the chosen treatment for the imaginal
discs is to heat the larvae at 47 °C for 15 min (Brower, Lawrence & Wilcox, 1981) while for
the flight muscles it is best to heat at 52 °C for 15 min (Lawrence, unpublished). The background may be due to other dehydrogenases and their substrates - after heat fixation these
substrates are probably free to diffuse out. An additional advantage of heating is that staining
times are reduced, probably because the succinate can diffuse in more easily. For example,
unheated eyes are best kept in the staining solution for several days; but after heating to 52 °C
for 10 min, the eyes stain well in about 12 h. Heat fixation does not seriously damage the
tissues. Freezing also removes the background but causes more damage.
(7) Temperature sensitivity of the lethal alleles
After chromosome 'cleaning', the chromosomes carrying sdh3, sdh4, sdh\ sdh6 and sdh*,
which are lethal at 25 °C, were tested to see if they were viable as homozygotes at 18 °C. All
trans combinations were also made at 18 °C and although most combinations were lethal it
was found that sdh8flieswere fully viable at 18 °C, as were sdh8[sdh*. A few sdh8/sdh5fliesalso
survive at 18 °C. Thus, sdh8 is a temperature-sensitive lethal; when transferred to 25 °C the
adults slowly sicken and die after about a week. At 25 °C sdh8fsdh6 first instar larvae hatch
normally but die before pupation, sdh8fliesgrown and kept at 17-18 °C are virtually male
sterile but female fertile.
A general cell marker for clonal analysis of Drosophila
325
6
The temperature-sensitive lethal, sdh , could be a useful tool: maternal perdurance of
succinate dehydrogenase or some precursor RNA might make clonal analysis of the embryo,
or even the larva, difficult or impossible. This might be overcome by breeding from sdhs
females reared at 18 °C and then transferring the progeny to 25 °C. This tactic has not yet
been tried.
RESULTS AND DISCUSSION
An ideal cell marker should be autonomous, so that all and only those cells
carrying the mutant genotype express the phenotype. The marker should be
gratuitous, so that even large areas of mutant tissue should develop and differentiate normally. The marker should have a short perdurance (Garcia-Bellido
& Merriam, 1971) so that it is visible even in small clones. Ideally the marker
should be scorable in the cells of embryos, larvae and adults and be expressed
in all cell types.
In the Material and Methods I describe the isolation of mutant alleles at
a locus affecting the activity of succinate dehydrogenase. These alleles are
homozygous lethal but viable in cells, where they reduce or eliminate succinate
dehydrogenase activity. In the following section some preliminary results with
this cell marker are reported. The tests are designed to see how far the sdh
phenotype approaches the ideal described above.
Fig. 1. Section through a M(2)S7+ sdh8 bw clone in the eye. The eye has been
stained for succinate dehydrogenase activity which gives the blue colour. The
red colour is due to the pigment cells in the wild-type parts of the eye. This eye
was heated to 49 °C for 10 min, stained for 24 h, embedded and sectioned at ca.
4 /tm. Note the clone is marked by the absence of both the red pigment in the pigment
cells and the blue staining in the cytoplasm and rhabdomeres of the retinula cells.
Larvae irradiated (1500R) at 44-52 h after egg laying. For a study of eye anatomy
and development see Ready, Hanson & Benzer (1976). x 230.
Fig. 2. Detail of section illustrated in Fig. 1 to show ommatidia mosaic for sdh
(open arrows) and sdh+ cells (closed arrows). The numbers refer to the particular
retinula cells. Note slight residual stain in rhabdomeres in the clone, and that they
are normally arranged. There is a clear difference between sdh and sdh+ cytoplasm
of the retinula cells but some of the rhabdomeres of those sdh cells which are close
to sdh+ cells stain darker blue, x 1000.
Fig. 3. Section through a sdh4 bw M(2)c+ clone in the eye. This was treated as the
eye in Fig. 1 and 2, but was not heated at all. Note, in comparison with Figs 1 and 2,
that the clone stains pale blue. Mosaic ommatidia can still be distinguished (m).
Larvae irradiated (1500R) at 48-72 h after egg laying, x 670.
Fig. 4. Section through en M(2)S7+ sdh8 bw clone in a en background. This eye was
heated to 53 °C for 15.min before staining. After this treatment all staining within the
clone is removed, and succinate dehydrogenase activity outside the clone is reduced
(compare with Fig. 1). Mosaic ommatidia can still be observed (arrows). Larvae
irradiated (1500R) at 72-96 h after egg laying, x 480.
Fig. 5. Section through eye of en sdh* bw/cn M(2)S7 genotype irradiated (1000R),
92-96 h after egg laying. The eye was heated to 52 °C for 15 min before staining.
One cell (retinula cell number 1, at the focus of the three arrows) is unstained and
presumably sdh. The arrangement of retinula cells (1-7) is shown in a nearby
ommatidium. x 1650. Oil immersion.
326
P. A. LAWRENCE
A general cell marker for clonal analysis of Drosophila
327
Cell autonomy of the marker
Markers are usually tested for autonomy by linking them genetically to
another marker and looking for cell-by-cell coexpression of the two phenotypes
(e.g. Janning, 1972). The eye was chosen for these tests, since the pigment and
retinula cells contain pigment which is largely removed by the brown mutation.
Larvae that were heterozygous for both brown and the marker (sdh3, sdh*, sdh6
and sdh8 have been tried so far) were irradiated and large Minute+ clones
produced. Flies bearing brown clones were found under the dissecting microscope, processed for succinate dehydrogenase staining and sectioned.
For each of the four alleles tested most clones show a clear phenotype both
in the pigment cells due to bw, and in the retinula cells due to sdh (Figs. 1-4).
A few clones show only the brown phenotype, presumably because the mitotic
recombination was distal to sdh but proximal to bw. Analysis of the bw sdh
clones show first, that cells in the ommatidia are normally arranged and
second, that the expression of the phenotype is autonomous, or nearly so.
When examined for succinate dehydrogenase staining using the light microscope, the border of the clone is sharp, but in spite of this it is difficult to score
unequivocally each retinula cell. This is so even in those sections where individual ommatidia are clearly a mosaic of sdh and sdh+ cells (Fig. 3). It is clear
that there is some spread of blue precipitate in the retinula cells; this is most
conspicuous in the rhabdomeres, but can be seen in the cytoplasm as well
(Fig. 3). Nevertheless the boundary of the clone is seen as a jagged interface
between dark-blue and pale-blue retinula cells. This boundary coincides very
well with the boundary between red (bw+) and pale-brown (bw) pigment cells
proving that the autonomy is good but not excluding the possibility of some
spread of stain across a cell or two. No more precise conclusion is possible,
because the retinula cells lose their red pigment during the long staining step,
and therefore cannot be independently scored for both the sdh and bw phenotypes.
We have found that heat fixation reduces non-specific precipitation of the
dye; therefore eyes were heated to 49, 52 or 53 °C before staining. This procedure
almost eliminates the background of pale-blue stain within the clone and
sharpens the clone border so that it becomes possible to score each retinula cell
Fig. 6. Electron microscope section through ommatidium at the border between
sdh bw clone and sdh+ bw+ background. The genotype of these cells can be identified
by the larger number of granules in the sdh+ bw+ cells (g); note the closely packed
endoplasmic reticulum in the sdh bw cells (4, 5) compared with the sdh+ bw+ cells
(1, 2, 3, 6 and 7). Note brown pigment cells (bw) in the upper part of the picture
are small and contain few granules while those below (bw+) are larger and filled with
granules (closed arrows). The sdh and sdh+ retinula cells shown are typical of those
found universally in the large sdh clone and sdh+ background, respectively,
x 17,500. Picture taken by Nichol Thomson.
328
P. A. LAWRENCE
A general cell marker for clonal analysis of Drosophila
329
unambiguously for sdh phenotype (Figs. 1, 2, 4). This result strongly argues for
cell-by-cell autonomy of the marker.
One eye bearing a large bw sdh clone was processed for the electron microscope (Ready et al. 1976). It was not heated or stained. The effect of bw can be
seen as a reduction in number of pigment granules (Fig. 6). The sdh cells are
distinct, their cytoplasm being condensed and darker than the sdh+ cells. The
endoplasmic reticulum is also more closely packed. This distinctive phenotype
is clear in retinula cells 1-6 but not in number 7 and is found in the bw area of
the eye. Along the zone where bw and bw+ territories meet, mosaic ommatidia
can be found (Fig. 6).
Additional evidence for cell autonomy comes from the appearance of clones
in the imaginal discs which have a sharp boundary (Fig. 7, and Brower et al.
1981). Moreover in clones within the adult epidermis, salivary glands (Fig. 8),
and gut (Fig. 9), individual cells can be scored. Further, following a suggestion
by Antonio Garcia-Bellido, some stw pwn sdhs M(2)c+ clones were examined
in newly emerged abdomens and thoraces (see Garcia-Bellido & Dapena (1974)
for a description of pawn). The overlying pattern of stw pwn trichomes in the
cuticle can be compared with the patch of sdh phenotype in the underlying
epidermal cells. I could not determine exactly which trichome was secreted by
which cell but the correlation between the mutant cells and mutant cuticle
appeared to be perfect. This confirms that the sdh marker is cell autonomous.
Is the marker gratuitous?
The overall appearance, size and shape of the eye clones is normal in young
adults, suggesting that they develop as is usual for Minute+ clones. Clones in the
head, leg and wing are of normal size and shape and respect compartment
boundaries. However, in older adults (from 2-3 days after emergence) sdh cells
in the eye slowly degenerate. Clones have so far been observed also in the adult
muscles, the gut, the salivary glands and the oenocytes and I have seen no other
phenotype than the lack of succinate dehydrogenase.
Fig. 7. sdh8 M(2)c+ clone in the wing (w) imaginal disc. Note that the mutant
(sdh8, open arrow) and wild-type (sdh+, closed arrow) cells meet at a sharp interface
(white arrow). Entire larvae were heated to 47 °C for 15 min before dissection and
staining of the disc (see Brower et al. 1981). The leg disc (1) is completely stained.
xl80.
Fig. 8. sdh6 M(2)c+ clone in adult salivary gland; note cells can be clearly scored
as sdh (open arrows) or sdh+ (closed arrows). 25 /tm wax section, x 440.
Fig. 9. sdh8 M(2)c+ clone in adult proventriculus (p). Note two sdh+ cells (closed
arrows at 2) which are surrounded by the sdh (open arrows) cells of the clone.
m, midgut; o, oesophagus. 25 fim wax section x 530.
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P. A. LAWRENCE
Perdurance
Perdurance is defined as the persistence in cells of wild-type phenotype after
loss of the wild-type gene (Garcia-Bellido & Merriam, 1971). A long perdurance
would reduce the usefulness of a marker mutation as small clones could not be
identified. It was therefore important to measure perdurance for a mutant allele
of sdh. Mutant cells were searched for in sections of eyes that had been irradiated
late in larval development and heated to 52 °C before staining. Small clones of
bw pigment cells are found and these are associated with retinula cells that do
not stain blue. In eyes from larvae irradiated at 96 h after egg laying, very small
clones could be detected, these are sometimes just one or two pigment cells. In
these eyes occasional unstained retinula cells are also seen. Unstained cells are
not found in eyes irradiated at earlier times and it is probable that these unstained cells are indeed small sdh clones (Fig. 5). Unfortunately, owing to the
loss of the red pigment in the retinula cells during staining, it was not possible
to decide if some sdh cells were overlooked or if some sdh+ cells were incorrectly
classified. Further, there is always the possibility that a marked retinula cell may
have been part of a slightly larger clone which included unscored cells such as
cone or bristle cells. Nevertheless the successful identification of a single sdh
retinula cell does suggest that perdurance is not a serious problem in the eye.
One would not expect perdurance of enzyme activity to be the same in all
tissues. It might depend on the number of cell divisions and on time elapsed
after removal of the sdh+ allele from the cell as well as other factors. In the case
of the eye, irradiation was at 96 h after egg laying but the eye was not fixed until
after adult emergence, some 5 days later.
How universal is the marker?
A proper answer to this question must await more study, but sdh clones have
so far been found in several different organs in the adult; the epidermis, the eye,
the flight and abdominal muscles, the heart, the oenocytes, the gut, and the
salivary glands. In sdh+ flies all cells contain mitochondria which precipitate the
stain, so it is conceivable that the marker may be useable in most cell types.
CONCLUSION
A new cell marker has been isolated and partially tested. The alleles affect the
activity of the enzyme succinate dehydrogenase. Clones of cells homozygous
for sdh8 are cell viable and usually normal, are autonomously expressed, and
are scorable in several different tissues. The perdurance, at least in the eye, is
such that clones of one or two cells can be scored. The marker thus looks
promising and would seem to be an improvement on existing enzyme markers
(see Hall et al. 1976).
A general cell marker for clonal analysis of Drosophila
331
I thank Brigid Hogan for a key conversation at Titisee in 1978. I am grateful to Paul
Johnston and Sheila Green who have done much of the work. The project would have
foundered without the good advice and persistent optimism of Gary Struhl who also helped
with the mutagenesis. I thank my colleagues at the MRC LMB and Michael Ashburner for
encouragement and criticism, and Philip Anderson for improving the manuscript.
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{Received 4 February 1981, revised 20 March 1981)